Calcium transport in the chick chorioallantoic membrane: Isolation and characterization of constituent cells, evidence for cellular compartmentalization, and techniques for the biochemical analysis of transport-related protein components

The chick eggshell supplies approximately 80% of the calcium found in the hatchling chick. The mobilization and transport of eggshell calcium into the developing embryo involve the transepithelial movement of large amounts of calcium across the ectodermal cells of the chorioallantoic membrane (CAM). A variety of microscopic, biochemical, and physiological techniques were used to prepare in vitro systems for the study of calcium transport. Isolated, cultured, CAM ectodermal cells which maintain characteristics of in ovo CAM calcium transport were prepared by selective purification. Preparations were characterized with respect to morphology, the expression of specific markers, and the maintenance of in ovo-like calcium uptake activity. Isolated CAM cells were used to characterize calcium accumulation by a combination of radioisotope uptake, Fura 2 fluorescence measurements of cytosolic calcium fluxes, and differential permeabilization of membrane with digitonin. Evidence from two separate sets of experiments is presented suggesting that calcium is sequestered away from the cytosol into endosome-like vesicles during uptake. The results presented here are consistent with a model for calcium transport involving endocytotic accumulation of calcium or sequestration of calcium into endosome-like structures after influx. In addition, two biochemical techniques are presented: (1) a discontinuous gel electrophoresis system, which allows the separation of proteins by molecular weight with the concomitant retention of native enzymatic activity, this method utilizes the cationic detergent cetyltrimethylammonium bromide and an arginine-Tricine buffer system, and (2) a rapid protein assay that combines the advantages of bicinchoninic acid based detection with a dramatic reduction in incubation time produced by 2.45 GHz microwave irradiation; accurate concentrations may be determined with 20 sec of microwave exposure. These two techniques should prove useful in the characterization of the protein components of CAM calcium transport and other proteins in general